The presence of chitinase activity in human serum has recently been described by us. On that occasion we speculated on the possible role of mammalian chitinases as a defense mechanism against chitin-containing pathogens. The results of the present study substantiate our hypothesis. We demonstrate and partially characterize the chitinase activities that are present in plasma of guinea pigs and in homogenates of A.fumigatus with the aid of the substrates MU-[GlcNAc]2,3 and also with glycol [3H]chitin. Upon infection with A.fumigatus the serum chitinase activity levels in the circulation of pathogen-free guinea pigs increased in a time-dependent manner. The increase was also dependent on the size of the infecting fungal inoculum. Antifungal treatment diminished the increases. The increased chitinase activity was of guinea pig origin. The activity of beta-hexosaminidase showed a very slight increase subsequent to the infection. The activities of three other enzymes of lysosomal origin (alpha-mannosidase, beta-galactosidase and beta-glucosidase) did not increase.

In recent studies the existence of a chitinase in various mammals, like man, was described. The aim of the present study was to find out whether salivas of periodontally healthy and inflamed humans also contain chitinase activity. Chitinase activity, assayed with the substrate 4-methylumbelliferyl-beta-D-N,N',N"-triacetylchitotrioside, was shown to be present in human whole saliva, with an activity level and apparent molecular mass (35 kDa) that were comparable with those of the human serum enzyme. Both lysozyme and beta-N-acetylhexosaminidase could be separated from chitinase by means of Bio-Gel P-100 gel filtration chromatography. The enzyme was also present in glandular saliva of parotid, palatine, submandibular and sublingual glands. The chitinase activity was not of oral epithelial, bacterial or plaque bacterial origin and was not correlated with the activity of salivary amylase. A comparative study of whole salivas of periodontally healthy controls and gingivitis and periodontitis subjects showed that only in the case of periodontitis there was a significant increase of the specific chitinase activity. The latter enzyme showed a gel filtration pattern that was comparable with that of the enzyme from controls. The measured albumin levels in saliva and the absence of correlation between the chitinase activity levels in plasma and saliva from periodontitis patients indicated that the (increased) chitinase activities did not originate from blood leakage to the oral cavity.

Intravenous infection of guinea pigs with the fungus Aspergillus fumigatus resulted in increased levels of chitinase in serum and tissues of the animals. The molecular properties of the enzyme were demonstrated to be different from those of the fungal chitinase, but also from guinea pig lysozyme and /?-Nacetylhexosaminidase. Bio-Gel P-100 gel filtration showed that in liver, spleen, heart and lung tissue of control animals there were two molecular mass forms present with apparent molecular masses of 35 kDa and 15 kDa. In brain and serum, only the 35 kDa form was detectable. Kidney showed only the 15 kDa form. Upon infection the 35 kDa form appeared in kidney and increased in the other tissues. When a less pathogenic form of the fungus was used the 35 kDa form remained absent in kidney. In contrast to human serum chitinase, the enzyme from guinea pig serum and tissues did bind to concanavalin ASepharose. This was the case for both molecular mass forms. The mode of cleavage of the substrate 4-methylumbelliferyl-tri-N-acetylchitotrioside (MU- [GlcNAc],, where GlcNAc is N-acetylglucosamine) by the two forms of the enzyme was the same: both [GlcNAc], and [GlcNAc], were released. The chitinase activity levels in the control tissues showed a large variation in this order: spleen > lung, kidney > liver > heart > brain. The fact that spleen showed the highest chitinase level is in agreement with its major role as a lymphoid organ in cases of systemic infections. The relative increases upon infection were the highest for the tissues that showed low control values.

Since 1988 an endoglucosaminidase, provisionally named MU-TACT hydrolase, has been known that hydrolyses the artificial substrate 4-methylumbelliferyl-tetra-N-acetyl-chitotetraoside (MU-[GlcNAc]4, where GlcNAc is N-acetylglucosamine). The biological function of the enzyme was unknown. In this paper evidence is presented showing that this endoglucosaminidase from human serum is in fact a chitinase that is different from lysozyme. The facts sustaining this finding are: (i) the identification of the products formed from MU-[GlcNAc]3 and [GlcNAc]2;and [GlcNAc]3; (ii) chitin and ethylene glycolchitin can be degraded by the enzyme; (iii) the chitinase inhibitor allosamidin also inhibits the action of MU-TACT hydrolase from human serum; (iv) no hydrolysis of the lysozyme substrate Micrococcus lysodeikticus. The enzyme also occurs in rat liver. It was demonstrated that upon Percoll density gradient centrifugation the enzyme from this tissue distributed parallel to the lysosomal marker enzymes beta-N-acetylhexosaminidase and beta-galactosidase, indicating a lysosomal localization for this enzyme. It is proposed that the enzyme functions in the hydrolysis of chitin, to which mammals are frequently exposed during infection by pathogens.

Human salivary chitinase could play a role in the defence against chitin-containing oral pathogens. The activity levels of chitinase in the whole saliva of periodontitis patients were significantly higher than those in saliva from controls. Periodontal treatment for a period of 5-6 months resulted in a three- to fourfold decrease in this enzyme activity. The activity of beta-N-acetylhexosaminidase, which is another enzyme that hydrolyses glycosidic linkages, also decreased as a result of treatment, although to a lesser extent. The decrease in chitinase activity upon treatment of the disease did not correlate with the decrease that was seen in clinical attachment loss and bleeding on probing, and only a weak correlation was observed with the changes in probing pocket depth and plaque index. No correlations were found between the above clinical parameters and the decrease in beta-N-acetylhexosaminidase activity.